Difference between revisions of "Part:BBa K3610044"

Latest revision as of 01:04, 28 October 2020

EFR ectodomain / SmallBit NanoLuc for C. reinhardtii

This part includes the ectodomain of the plant pattern recognition receptor EFR fused to the sequence for the SmallBit of the split-NanoLuc protein. The sequence for the receptor and the SmallBit protein have been codon optimized for expression in C. reinhardtii. To ensure localization at the membrane, this part further contains the sequence for the signal peptide SP7 from C. reinhardtii and the self-cleaving protein from the foot-and-mouth disease (FMD) virus.

Usage and Biology

EFR

Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants first defence mechanism against potential pathogens. The EFR receptor is also a leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.

Usage with NanoLuc

In this case, the C-terminal domain of EFR, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.

The ligand-dependent interaction of EFR with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. We were interested in testing, whether coexpression with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase will allow elf18-induced interaction between BAK1 and EFR, which then could drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output, which would allow the visualization of the ligand-dependent interaction between EFR and BAK1.

This gives this part the potential to be used, in coordination with the BAK1 ectodomain and mCherry, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a funcitonal protein, which gives a visual uotput with the substrate furimazine.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 93
Illegal NheI site found at 235
Illegal NheI site found at 1165
Illegal NheI site found at 2083
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 148
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1751
1000
COMPATIBLE WITH RFC[1000]

@@ Line 12: / Line 12: @@
 In this case, the C-terminal domain of EFR, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.
-The ligand-dependent interaction of EFR with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither EFR nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, elf18-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR and BAK1.
+The ligand-dependent interaction of EFR with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain.
-This enables us to use this part, in coordination with the BAK1 ectodomain and mCherry, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a funcitonal protein, which gives a visual uotput with the substrate furimazine.
+We were interested in testing, whether coexpression with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase will allow elf18-induced interaction between BAK1 and EFR, which then could drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output, which would allow the visualization of the ligand-dependent interaction between EFR and BAK1.
+This gives this part the potential to be used, in coordination with the BAK1 ectodomain and mCherry, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a funcitonal protein, which gives a visual uotput with the substrate furimazine.
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